This study aimed to determine whether aging negatively affects MSC replication and osteogenesis and whether these features could be altered by exposure to an extracellular matrix (ECM) generated by marrow cells from young or old mice. A cell-free ECM was prepared from cultured femoral marrow cells from either 3- or 18-mo-old C57BL/6 mice (young-ECM or old-ECM, respectively). The replication and osteogenesis of young or old MSCs maintained on young-ECM vs. old-ECM as well as plastic were examined in vitro and in vivo. We found that the frequency of MSCs in marrow from old mice, measured by colony-forming cells, was only marginally lower than that of young mice. In contrast, defects in the self-renewal and bone formation capacity of old MSCs were remarkable. These defects were corrected by provision of a young-ECM but not old-ECM. In parallel cultures maintained on a young-ECM, the intracellular levels of reactive oxygen species from both old and young mice were reduced 30-50% compared to those maintained on old-ECM or plastic. We concluded that aging negatively affects the formation of an ECM that normally preserves MSC function, and aged MSCs can be rejuvenated by culture on a young-ECM.
"The aging process itself has been associated with defective ECM production. For example, the reduced self-renewal and bone formation capacity of aged mesenchymal stem cells (MSCs) was corrected by culturing them on ECM from young MSCs (Sun et al. 2011). In analogy to that, proliferative defects of mouse adult fibroblasts harboring HGPS-linked Lmna mutation were rescued upon growth on ECM derived from wild-type cells (Hernandez et al. 2010). "
"In contrast, bone formation capacity of cells expanded on tissue culture plastic was dramatically diminished after 6e7 passages. Also, Sun et al. has shown that culturing freshly isolated human bone marrow mononuclear cells on stromal cell-derived extracellular matrix enhances the formation of colonies comprised of either osteoblast-like, fibroblast-like, or adiopcyte-like cells . In addition, they also showed that culturing late-passage MSCs on fresh ECM recovered or at least retained the desired properties in these older cells. "
[Show abstract][Hide abstract] ABSTRACT: Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and "stemness". Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼1.6×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼2.2×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.
"Studies in different tissues across species have clarified that the ECM constitutes an important part of progenitor cell niches [7,8]. While certain microenvironments in Drosophila, as expected, repress stem cell differentiation and promote adherence to the niche , even age-related differences in ECM composition directly influence stem cell function . To begin understanding the remodeling events in the ECM microenvironment when HPCs are activated in a second tier of defense to liver injury, we used the AAF/PHx protocol to study transcriptional changes in ECM constituents during three phases of an HPC response, that is, the activation phase, the early proliferation and migration phase, and the late proliferation and migration phase. "
[Show abstract][Hide abstract] ABSTRACT: Tissue repair in the adult mammalian liver occurs in two distinct processes, referred to as the first and second tiers of defense. We undertook to characterize the changes in molecular constituents of the extracellular matrix when hepatic progenitor cells (HPCs) respond in a second tier of defense to liver injury.
We used transcriptional profiling on rat livers responding by a first tier (surgical removal of 70 % of the liver mass (PHx protocol)) and a second tier (70 % hepatectomy combined with exposure to 2-acetylaminofluorene (AAF/PHx protocol)) of defense to liver injury and compared the transcriptional signatures in untreated rat liver (control) with those from livers of day 1, day 5 and day 9 post hepatectomy in both protocols. Numerous transcripts encoding specific subunits of collagens, laminins, integrins, and various other extracellular matrix structural components were differentially up- or down-modulated (P < 0.01). The levels of a number of transcripts were significantly up-modulated, mainly in the second tier of defense (Agrn, Bgn, Fbn1, Col4a1, Col8a1, Col9a3, Lama5, Lamb1, Lamb2, Itga4, Igtb2, Itgb4, Itgb6, Nid2), and their signal intensities showed a strong or very strong correlation with Krt1-19, a well-established marker of a ductular/HPC reaction. Furthermore, a significant up-modulation and very strong correlation between the transcriptional profiles of Krt1-19 and St14 encoding matriptase, a component of a novel protease system, was found in the second tier of defense. Real-time PCR confirmed the modulation of St14 transcript levels and strong correlation to Krt-19 and also showed a significant up-modulation and strong correlation to Spint1 encoding HAI-1, a cognate inhibitor of matriptase. Immunodetection and three-dimensional reconstructions showed that laminin, Collagen1a1, agrin and nidogen1 surrounded bile ducts, proliferating cholangiocytes, and HPCs in ductular reactions regardless of the nature of defense. Similarly, matriptase and HAI-1 were expressed in cholangiocytes regardless of the tier of defense, but in the second tier of defense, a subpopulation of HPCs in ductular reactions co-expressed HAI-1 and the fetal hepatocyte marker Dlk1.
Transcriptional profiling and immunodetection, including three-dimensional reconstruction, generated a detailed overview of the extracellular matrix constituents expressed in a second tier of defense to liver injury.
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